TY - JOUR
T1 - Inducing pluripotency in somatic cells from the snow leopard (Panthera uncia), an endangered felid
AU - Verma, Rajneesh
AU - Holland, Michael
AU - Temple-Smith, Peter
AU - Verma, Paul
PY - 2012
Y1 - 2012
N2 - Induced pluripotency is a new approach to produce embryonic stem-like cells from somatic cells that provides a unique means
to understand both pluripotency and lineage assignment. To investigate whether this technology could be applied to endangered
species, where the limited availability of gametes makes production and research on embryonic stem cells difficult, we attempted
generation of induced pluripotent stem (iPS) cells from snow leopard (Panthera uncia) fibroblasts by retroviral transfection with
Moloney-based retroviral vectors (pMXs) encoding four factors (OCT4, SOX2, KLF4 and cMYC). This resulted in the formation
of small colonies of cells, which could not be maintained beyond four passages (P4). However, addition of NANOG, to the
transfection cocktail produced stable iPS cell colonies, which formed as early as D3. Colonies of cells were selected at D5 and
expanded in vitro. The resulting cell line was positive for alkaline phosphatase (AP), OCT4, NANOG, and Stage-Specific
embryonic Antigen-4 (SSEA-4) at P14. RT-PCR also confirmed that endogenous OCT4 and NANOG were expressed by snow
leopard iPS cells from P4. All five human transgenes were transcribed at P4, but OCT4, SOX2 and NANOG transgenes were
silenced as early as P14; therefore, reprogramming of the endogenous pluripotent genes had occurred. When injected into
immune-deficient mice, snow leopard iPS cells formed teratomas containing tissues representative of the three germ layers. In
conclusion, this was apparently the first derivation of iPS cells from the endangered snow leopard and the first report on induced
pluripotency in felid species. Addition of NANOG to the reprogramming cocktail was essential for derivation of iPS lines in this
felid. The iPS cells provided a unique source of pluripotent cells with utility in conservation through cryopreservation of genetics,
as a source of reprogrammed donor cells for nuclear transfer or for directed differentiation to gametes in the futur
AB - Induced pluripotency is a new approach to produce embryonic stem-like cells from somatic cells that provides a unique means
to understand both pluripotency and lineage assignment. To investigate whether this technology could be applied to endangered
species, where the limited availability of gametes makes production and research on embryonic stem cells difficult, we attempted
generation of induced pluripotent stem (iPS) cells from snow leopard (Panthera uncia) fibroblasts by retroviral transfection with
Moloney-based retroviral vectors (pMXs) encoding four factors (OCT4, SOX2, KLF4 and cMYC). This resulted in the formation
of small colonies of cells, which could not be maintained beyond four passages (P4). However, addition of NANOG, to the
transfection cocktail produced stable iPS cell colonies, which formed as early as D3. Colonies of cells were selected at D5 and
expanded in vitro. The resulting cell line was positive for alkaline phosphatase (AP), OCT4, NANOG, and Stage-Specific
embryonic Antigen-4 (SSEA-4) at P14. RT-PCR also confirmed that endogenous OCT4 and NANOG were expressed by snow
leopard iPS cells from P4. All five human transgenes were transcribed at P4, but OCT4, SOX2 and NANOG transgenes were
silenced as early as P14; therefore, reprogramming of the endogenous pluripotent genes had occurred. When injected into
immune-deficient mice, snow leopard iPS cells formed teratomas containing tissues representative of the three germ layers. In
conclusion, this was apparently the first derivation of iPS cells from the endangered snow leopard and the first report on induced
pluripotency in felid species. Addition of NANOG to the reprogramming cocktail was essential for derivation of iPS lines in this
felid. The iPS cells provided a unique source of pluripotent cells with utility in conservation through cryopreservation of genetics,
as a source of reprogrammed donor cells for nuclear transfer or for directed differentiation to gametes in the futur
UR - http://www.ncbi.nlm.nih.gov/pubmed/22079579
U2 - 10.1016/j.theriogenology.2011.09.022
DO - 10.1016/j.theriogenology.2011.09.022
M3 - Article
SN - 0093-691X
VL - 77
SP - 220
EP - 228
JO - Theriogenology
JF - Theriogenology
IS - 1
ER -